WO2022129651A1

WO2022129651A1 - Tyrosinase-inhibiting molecules and dermopharmaceutical composition that includes them

Info

Publication number: WO2022129651A1
Application number: PCT/ES2021/070225
Authority: WO
Inventors: Alfredo MARTÍNEZ GUTIÉRREZ; Alexandra BERTRAN JUNQUÉ; María del Carmen GONZÁLEZ RODRÍGUEZ; Sergio PASCUAL DEL PRADO; Luís Shotze LUIS GARCÍA
Original assignee: Mesoestetic Pharma Group, S.L
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2022-06-23
Also published as: ZA202307066B; EP4223740A1; CO2023012852A2; AU2021403876A1

Abstract

The present invention provides tyrosinase-inhibiting molecules of general formula (I) and a dermopharmaceutical or cosmetic composition that includes at least one of said tyrosinase-inhibiting molecules.

Description

DESCRIPTION

TYROSINASE INHIBITOR MOLECULES AND DERMOPHARMACEUTIC OR COSMETIC COMPOSITION THAT INCLUDES THEM

The present invention relates to new tyrosinase inhibitor molecules, as well as to a dermopharmaceutical or cosmetic composition that includes at least one of said tyrosinase inhibitor molecules.

More specifically, in a first aspect, the invention provides new tyrosinase inhibitor molecules with the following general formula (I):

where R ₁ and R ² are selected, independently of each other, from H, HSO ₃ or one of their salts with a physiologically acceptable cation and represents a single or double bond, where R ¹ and R ² do not both represent H. which have a high solubility in water compared to other molecules that share the same structure.

In a second aspect, the invention provides a dermopharmaceutical or cosmetic composition comprising at least one tyrosinase inhibitor molecule of general formula (I) as described above in combination with suitable excipients for its formulation.

Skin pigmentation disorders consisting of hyperproduction of melanins or hypermelanosis are common. Hypermelanosis appear more frequently in certain periods of life and are usually responsible, among others, for exogenous factors, such as excessive exposure to sunlight. These hypermelanoses they are characterized by the appearance on the skin, particularly in uncovered areas, of dark and colored spots that give it a certain degree of visual heterogeneity; It is usually an aesthetic problem. These spots are caused by melanocytic hypertrophy or by a greater accumulation of melanins in the keratinocytes located in the superficial part of the epidermis. Likewise, various dysfunctions of melanogenesis due to the effect of exogenous aggressions, for example hormonal alterations or aging, cause the appearance of spots of melanic origin, brown, mamon or blackish in color, in the form of melasmas, ephelides, senescence spots, lentigines , etc.

Thus, for example, melasma is a topical condition that consists of an increase in pigmentation in the form of irregular and asymmetric macules in localized areas of the skin, mainly in areas exposed to the sun. It is an acquired hypermelanosis characterized by asymmetric, brownish, irregular and reticulated macules on sun-exposed areas of the skin, especially on the face. To date, the pathogenesis of melasma has not been fully elucidated; However, chronic ultraviolet (UV) exposure, female hormonal stimulation, and genetic background have been proposed to play a role in the development of melasma (O. A. Ogbechie-Godec, N. Elbuluk. Dermatol Ther (Heidelb) 2017 , 7(3), 305). The management of melasma is a great challenge, especially since it is prone to frequent relapses (Kwon SH, et al. Melasma: Updates and perspectives. Exp Dermatol. 2019; 28(6): 704-708).

In the pigmentation process, the enzyme tyrosinase is key in the synthesis of melanin, and tyrosinase inhibitors are a widely used approach to treat hyperpigmentary conditions (Zolghadri S, et al. A comprehensive review on tyrosinase inhibitors. J Enzyme Inhib Med Chem 2019;34(1):279-309). Thus, since tyrosinase plays a key role in the process of melanin production, inhibitors of this enzyme are often used as skin depigmenting agents.

EP 1857109 A2, for example, describes phenylthiourea derivatives as inhibitors of mammalian tyrosinase in acellular extracts of mammalian melanocytes or melanoma cells. EP 2117498 B1 discloses 4-hydroxyphenoxyacetic acid derivatives for skin lightening and/or whitening and/or for pigmentation reduction and/or for hyperpigmentation reduction and/or for melanogenesis inhibition . Among the different chemical families that act on this enzyme, chalcones have shown promising results, exerting a powerful inhibitory activity on tyrosinase (Kostopoulou I, et al. Recent Developments on Tyrosinase Inhibitors based on the Chaicone and Aurone Scaffolds. Current Enzyme Inhibition 2018;14:3-17;Khatib S, et al.Chaicones as potent tyrosinase inhibitors: the importance of a 2,4-substituted resorcinol moiety.Bioorg Med Chem. 2005;13(2):433-41).

However, these molecules known to be tyrosinase inhibitors are highly insoluble in water, which limits their use in cosmetic formulations.

In this context, it would be essential to find molecules that are effective in inhibiting tyrosinase and that are also water soluble and therefore suitable for use in skin care products.

Thus, the objective of the present invention is to find molecules that meet these conditions based on the efficacy of chalcones as depigmenting agents.

Starting from the substructure of the chalcone family and the position of the hydroxyl groups, since it has been shown that the location of these hydroxyl groups is key in the inhibition of tyrosinase by chalcones (Nerya O, et al. , "Chalcones as potent tyrosinase inhibitors: the effect of hydroxyl positions and numbers". Phytochemistry. 2004; 65(10): 1389-95), the present invention provides new tyrosinase inhibitor molecules with the following general formula (I):

where R ¹ and R ² are independently selected from H, HSO ₃ or one of their salts with a monovalent cation, M^SO3-, or with any physiologically acceptable cation, and

represents a single or double bond, with the proviso that R ¹ and R ² do not both represent H simultaneously, which have a high solubility in water compared to other molecules that share the same structure and, therefore, are useful for use as depigmentants in skin products.

In one embodiment, in the molecule of general formula (I), R ¹ and R ² are both HSO ₃ and is a double bond, according to formula (II):

In another embodiment, in the molecule of general formula (I), where one of R ¹ or R ² is HSO ₃ , and the other of R ¹ and R ² is H and

is a double bond, according to formulas (III):

In another embodiment, in the molecule of general formula (I), R ¹ and R ² are both M ⁺ SO ₃ ' and is a single bond, according to formula (IV):

In a preferred embodiment of the compound of formula (IV), M ⁺ is a sodium, potassium or triethylammonium cation.

Likewise, in another embodiment, in the molecule of general formula (I), R ¹ is M ⁺ SO ₃ - and R ² is H, it is a double bond, according to formula (V):

In a preferred embodiment of the compound of formula (V), M is a sodium, potassium or triethylammonium cation.

In a final embodiment, in the molecule of general formula (I), R ¹ and R ² are both M ⁺ SO ₃ - and is a double bond, according to formula (VI):

In a preferred embodiment of the compound of formula (VI), M is a sodium, potassium or triethylammonium cation.

According to the second aspect of the invention, a dermopharmaceutical or cosmetic composition is provided that comprises at least one tyrosinase inhibitor molecule of general formula (I) as described above in combination with suitable excipients for its formulation.

In this context, embodiments of the dermopharmaceutical or cosmetic composition of the invention include the tyrosinase inhibitory molecules of formula (I) or of formulas (II) to (VI), alone or in combination of at least two of them, together with suitable excipients for their formulation.

Preferably, the tyrosinase inhibitor molecule is present in the dermopharmaceutical or cosmetic composition in a concentration of 0.001-25% by weight.

As mentioned above, the dermopharmaceutical or cosmetic composition of the invention includes suitable excipients for its formulation for administration. Such excipients can be selected from oily solvents, anhydrous solvents, hydroalcoholic solvents, aqueous solvents, fillers, preservatives, gelling agents, perfumes, surfactants, sunscreens, antioxidants, viscosifiers, emulsifiers, silicones, as well as any of the ingredients used in the field. cosmetic or dermatological, in the usual concentrations of use, provided that said excipients do not interfere with the tyrosinase inhibitory activity of the combination described.

The composition of the invention can be presented in any galenic or cosmetic form of those usually used for application, for example in the form of an aqueous, hydroalcoholic, hydroglycolic or oily solution. It can also be included in oil-in-water or water-in-oil emulsions or multiple emulsions or formulated as a serum, foam or spray.

The invention is illustrated below by means of the following examples, which are illustrative and not limiting of the same and with reference to the attached figures, in which:

Fig. 1: Graph showing the tyrosinase activity, in percentage, of the molecules of formulas (II) to (VI) at a concentration of 1mM.

Fig. 2: Graph showing the percentage decrease in melamine content in samples of human melanoids treated with the molecules of formulas (II) and (III).

Efficacy of the molecules of general formula (I)

Tyrosinase inhibition To evaluate the possible inhibition of tyrosinase by these molecules, a computational model of human tyrosinase was initially developed based on known structures of tyrosinases from other organisms, since human tyrosinase has not crystallized and, therefore, is not available. of its actual three-dimensional structure.

This computational model was used in order to perform a virtual assessment of the affinity of different structures of formula (I). A theoretical solubility study using logP was also carried out in parallel with a study of the synthetic viability of each of them.

The results obtained are shown in Table 1 below.

Given the results, an in vitro assay was performed on human meianocytes. To stimulate melanogenic activity in the cells, they were treated with L-tyrosine. This compound, in addition to being a substrate for the enzyme tyrosinase, which is key in melanin synthesis, is capable of activating cell receptors that activate the signaling pathways that regulate melanogenesis (Slomínskí A, et al. L-tyrosine and L- dihydroxyphenylalanine as hormone-like regulators of melanocyte functions.Pigment Cell Melanoma Res.2012;25(1):14-27). With this condition, it is intended to stimulate melanogenesis in meianocytes with the aim of extracting tyrosinase and testing the effect of the molecules of the invention.

Thus, human meianocytes were cultured in culture medium (#PCS-200-013, ATCC) and L-tyrosine (2 mM) for 3 days. After the stimulation treatment, the cells were trypsinized and used in PBS pH 7.0 with 1% Triton X-100. The cell lysate, which contains tyrosinase, was incubated together with the molecules of the invention at different concentrations (0.1 and 1 mM), L-DOPA and MBTH (Winder AJ and Harris H. New assays for tile tyrosine hydroxylase and dopa oxidase activities of tyrosinase Eur J Biodiem 1991;198(2): 317-26). L-DOPA acts as a substrate for tyrosinase in the enzymatic reaction, and MBTH is a compound that binds to the hydroquinone formed by the oxidation of L-DOPA, generating a color complex. Each of the conditions used was prepared in triplicate. Finally, the absorbance at 492 nm was monitored using a spectrophotometer every 10 min for 2-5 h.

The results obtained are shown in table 2 and figure 1. The control stimulated with L-tyrosine is considered 100% to establish a reference value. In the case of the samples treated with the different molecules, molecule (III) is the one with the lowest tyrosinase activity (82% at 0.1 mM and 44% at 1 mM) followed by the molecule of formula (II) (97% at 0.1 mM and 55% at 1 mM). In the presence of the molecules of formulas (IV) to (VI), the tyrosinase activity is reduced (97%, 82%, 83% and 91% respectively, at 1 mM), but to a lesser extent compared to the first two molecules.

Table 1. Percentage of tyrosinase activity of the synthesized molecules at two different concentrations (0.1 and 1 mM).

Melanin inhibition

In order to study the inhibition of melanin molecules, an in vitro assay was performed on human melanocytes using conditions similar to those described in the tyrosinase inhibition assay. L-tyrosine is used to stimulate melanogenic activity in cells in order to extract melanin. Here only the molecules with the best inhibition against tyrosinase activity (formulas (II) and (III)) were tested. In this experiment, the concentration of the molecules used is the highest possible taking into account the value of dtotoxicity in melanocytes, 1.25 mM for the molecule of formula (II) and 0.33 mM for the molecule of formula (III). Thus, human melanocytes are cultured in culture medium (#PCS-200-013, ATCC). L-tyrosine (2 mM) was added along with the treatment molecules. All conditions were prepared in triplicate. After incubating the cells with the treatments for 3 days, they were trypsinized and lysed in 1M NaOH. The absorbance of cell lysates was measured at 340 nm using a spectrophotometer. The absorbance value is directly proportional to the melanin content.

As seen in figure 2, which shows the melarana content of the prepared samples, the control stimulated with L-tyrosine is considered 100% to establish a reference value. The difference between the melanin content of the samples treated with the molecules of formulas (II) (78 ± 7%) and (III) (49 ± 7%) and the sample treated only with L-tyrosine (100 ± 3%) is significant, according to Student's t-test.

Solubility of synthesized molecules

In order to study and compare the solubility of molecules in water, the following test is performed.

For each of the molecules, solutions are prepared in DMSO and in water. All samples are prepared at a final concentration of 50 mg/ml. After one hour of stirring at room temperature, the samples are filtered and HPLC is performed.

To calculate the percentage of solubility of each one of the molecules, the area of the peak obtained by HPLC of the sample dissolved in water and in DMSO is determined. The results are given in percentage of solubility of the compound dissolved in water between the compound dissolved in DMSO.

Table 3 shows the results obtained. It is observed that the reference molecule is completely insoluble in water at a concentration of 50 mg/ml. Molecules (II) and (III) are very soluble at this concentration and molecules of formulas (IV) to (VI) are completely soluble in water at the concentration tested.

Table 2. Percentage solubility in water compared to samples dissolved in DMSO

Synthesis of the molecules of the invention Formula (II): 5-[(E)-2-(4-hydroxy-3-sulfdbenzoyl)-1-ethenyl]-2-hydroxybenzenesulfonic acid

Under a nitrogen atmosphere, 1.039 g (4.33 mmol) of (E)-1,3-bis(p-hydroxyphenyl)-2-propen-1-one were slowly added to 5.04 g (43.3 mmol) of chlorosulfonic acid at 0 °C. The reaction was kept at room temperature for 22 h and the resulting mixture was slowly added to 25 ml of cold water. The obtained aqueous tese was extracted twice with 20 ml of ethyl acetate, the combined organic phases were washed with 10 ml of water and concentrated to dryness, to obtain 2.017 g of a solid containing áddo 5-[(E)- 2-(4-hydroxy-3-sulfobenzoyl)-1-ethenii]-2-hydroxybenzenesulfonic acid (86.3% HPLC area).

1H NMR (d-DMSO, 360 MHz): 6.86 (d, 1H), 6.91 (d, 1H), 7.62 (d, 1H), 7.70 (d, 1H), 7, 82 (d, 1H), 7.86 (s, 1H), 8.14 (s, 1H), 8.21 (s, 1H).

Formula (ill): 5-((E)-3-(p-hydroxyphenyl)acryloyl]-2-hydroxybenzenesulfonic acid

5-Acetyl-2-hydroxybenzenesulfonic acid (synthetic intermediate)

2 g (14.69 mmol) of 1-(p-hydroxyphenii)-1-ethanone was slowly added to 17.6 g (146.9 mmol) of chlorosulfonic acid at 0 °C. After 18 h at room temperature, 75 mL of cold water was added to the mixture, and the mixture was extracted twice with 50 mL of ethyl acetate. The organic phase was concentrated to dryness under vacuum to obtain 1.95 g of crude product containing 1.71 g (54% yield) 5-acetyl-2-hydroxybenzenesulfonic acid and 0.24 g of 1-(p-hydroxyphenyl )-1-ethanone.

1H NMR (d-DMSO, 360 MHz): 62.50 (3H), 6.88 (d, 1H), 7.84 (d, 1H), 8.06 (s, 1H).

5-[(E)-3-(p-hydroxyphenyl)acryloyl]-2-hydroxybenzenesulfonic acid

1.61 ml (26 mmol) of 98% sulfuric acid were added slowly to a mixture of 563 mg (2.60 mmol) of the crude product above and 362 mg (2.60 mmol) of p-hydroxybenzaldehyde in 6.4 mL of ethanol at 0 °C. The reaction was kept at room temperature for 21 h and the obtained mixture was slowly added to a solution of 2 g sodium chloride in 10 ml of water at 0 °C. The suspension was extracted twice with 20 ml of ethyl acetate and the combined organic phases were treated with anhydrous sodium sulfate and concentrated to dryness. The product obtained was purified on a silica gel column eluting with a gradient 100% didoromethane to didoromethane/methanol 8:2 to obtain 230 mg (yield 27.6%) of áddo 5-[(E)-3-(p- hydroxyphenyl)acryloyl)-2-hydroxybenzenesulfonic acid.

1H NMR (d-DMSO, 360 MHz): 66.80-6.88 (2H), 6.91-6.97 (1H), 7.61-7.69 (2H), 7.69-7, 78 (1H), 8.06-8.15 (1H), 8.18-8.25 (1H).

hydroxybenzenesulfonate, dipotassium salt

Under a nitrogen atmosphere, 20.8 g (86.63 mmol) of (E)-1,3-bis(p-hydroxyphenyl)-2-propen-1-one were slowly added to 138.25 g (1.18 mol) of chlorosulfonic acid at 0 °C. The reaction was kept at room temperature for 19 h and the resulting mixture was slowly added to 650 ml of cold water. The aqueous phase obtained was extracted twice with 500 ml of ethyl acetate, the combined organic phases were washed with 250 ml of water and concentrated to dryness to obtain 60.66 g of a product containing 66% 5-[ (E)-2-(4-hydroxy-3-sulfobenzoyl)-1-ethenyl]-2-hydroxybenzenesulfanic acid. 60.1 g of this product was suspended in 230 ml of water and an equimolecular amount of potassium carbonate was slowly added to obtain a complete solution. 600 ml of 2-propanol was added to obtain a suspension. The solid was filtered, washed twice with 200 ml of 2-propanol and dried under vacuum at 100 °C to constant weight, to obtain 19 g (yield 46%) of dipotassium salt of 5-{(E)-2 -(4-hydroxy-3-sulfonatobenzoyl)-1-ethen¡l}-2-hydroxybenzenesulfonate.

1H NMR (d-DMSO, 360 MHz): 6.86 (d, 1H), 6.91 (d, 1H), 7.63 (d, 1H), 7.71 (d, 1H), 7, 82 (d, 1H), 7.87 (s, 1H), 8.15 (s, 1H), 8.22 (s, 1H), 10.98 (brs, 1H), 11.18 (brs, 1H ).

Formula (V) (M = Na): sodium 5-[(E)-3-(p-hydroxyphenyl)acryloyl]-2-hydro)dbenzenesulfonate

1 g of 5-[(E)-3-(4-hydroxyphenyl)acryloyl]-2-hydroxybenzenesulfonic acid (3.12 mmol) was dissolved in 10 mL of 2-propanol and an equimolar amount of sodium bicarbonate was added slowly. The solid was filtered, washed twice with 2 ml of 2-propanol and dried under vacuum at 50 °C to constant weight, obtaining 284 mg (27% yield) of 5-[(E)-3-(p- sodium hydroxyphenyl)acryloylj-2-hydroxybenzenesulfonate.

1H NMR (d-DMSO, 360 MHz): 66.80-6.88 (2H), 6.91-6.97 (1H), 7.61-7.69 (2H), 7.69-7, 78 (1H), 8.06-8.15 (1H), 8.18-8.25 (1H), 10.98 (brs, 1H).

Formula (V) (M = (C ₂ H ₅ ) ₃ N): Triethylammonium 2-hydroxy-5-[2-(4-hydroxy-3-sulfonatobenzoyl)ethyl]- benzenesulfonate

5-[(E)-3-(4-hydroxyphenyl)acryloyl]-2-hydroxybenzenesulfonic acid (31.23 mmol) was dissolved in 70 mL of 2-propanol and 13.05 mL of trimethylamine ( 93.69mmol). The solid was filtered, washed twice with 20 ml of 2-propanol and dried under vacuum at 50 °C until constant weight, obtaining 10.6 g (81% yield) of 5-[(E)-3-( Triethylammonium p-hydroxyphenyl)acryloyl)-2-hydroxybenzenesulfonate.

1H NMR (d-DMSO, 360 mHz): 6 1.06 (t, 9H), 2.80 (q, 6H), 6.83 (d, 2H), 6.93 (d, 1H), 7, 66 (s, 2H), 7.73 (d, 2H), 8.09 (dd, 1H), 8.21 (d, 1H). Formula (Vi) (M = K): 2-hydroxy-5-[2-(4-hydroxy-3-sulfonatobenzoyl)ethyl)benzenesulfonate, dipotassium salt

In a high pressure reactor, 1 g (2.10 mmol) of 5-[(E)-2-(4-hydroxy-3-sulfonatobenzoyl)-1-ethenyl]-2-hydroxyphencenesulfonate, dipotated salt, was dissolved in 10 ml of water. 150 mg of Pd/C 10% were added and the mixture was hydrogenated at atmospheric pressure and 1500 RPM for 72 h at room temperature. The reaction was monitored by 1H NMR. The reaction mixture was filtered through a pad of Celite® and concentrated to dryness to give 2-hydroxy-5-(2-(4-hydroxy-3-sulfonatobenzoyl)ethylbenzenesulfonate dipotassium salt.

1H NMR (d-DMSO, 360 MHz): 6 2.80 (t, 2H), 3.20 (t, 2H), 6.66 (d, 1H), 6.86 (d, 1H), 7, 10 (d, 1H), 7.32 (s, 1H), 7.89 (d, 1H), 8.08 (s, 1H), 10.36 (brs, 1H), 11.12 (brs, 1H ).

Reference molecule: (E)-1,3-bis(p-hydroxyphenyl)-2-propen-1-one

To a mixture of 2 g (16.38 mmol) of p-hydroxybenzaldehyde and 2.23 g (16.38 mmol) of 1-(p-hydroxyphenyl)-1-ethanone in 20 ml of ethanol, at room temperature, slowly added 16.06 g (164 mmol) of 98% sulfuric acid. The reaction was kept at room temperature for 23 h and the mixture obtained was slowly added to a solution of 10 g of sodium chloride in 33 ml of water at 0 °C. The suspension was extracted three times with 16 ml of ethyl acetate and the combined organic phases were washed twice with 16 ml of water. After treating with anhydrous sodium sulfate, the organic phase was concentrated in vacuo to dryness to obtain 3.58 g (91% yield, 97.4% HPLC area) of (E>-1,3-bis(p-hydroxyphenyl )-2-propen-1-one.

1H NMR (d-DMSO, 360 mHz): 66.33 (d, 1H), 6.71 (d, 2H), 7.45 (s, 2H), 7.51 (d, 2H), 7.76 (dd, 1H), 8.21 (d, 1H).

Example of compositions of the invention in cream form

Example of compositions of the invention in serum form

Claims

1. Tyrosinase inhibitor molecules of the following general formula (I):

where R ¹ and R ² are selected, independently of each other, from H, HSO ₃ or one of their salts with a monovalent cation, M ⁺ SO ₃ ", or with any physiologically acceptable cation, and represents a single or double bond, with the

provided that R ¹ and R ² do not both represent H simultaneously.

2. Tyrosinase inhibitor molecules according to claim 1 selected from the following formulas (II) to (VI): II R ¹ and R ² are both HSO ₃ and

is a double bond

one of R ¹ or R ² is HSO ₃ , and the other of R ¹ and R ² is H and

is a double bond

IV R ¹ and R ² are both M ⁺ SO ₃ - and it is a single bond

VR ¹ is M ⁺ SO ₃ -and R ² is H,

is a double bond

VI R ¹ and R ² are both M ⁺ SO ₃ - and it is a double bond

3. Tyrosinase inhibitor molecules according to claim 1 or 2, where M is sodium, potassium or triethylammonium.

Dermopharmaceutical or cosmetic composition comprising at least one tyrosinase inhibitor molecule of general formula (I) according to claim 1, in combination with suitable excipients for its formulation.

5. Dermopharmaceutical or cosmetic composition comprising at least one tyrosinase inhibitor molecule selected from formulas (II) to (VI) according to claim 2, in combination with suitable excipients for its formulation.

6. Dermopharmaceutical or cosmetic composition according to claims 4 or 5, wherein the tyrosinase inhibitor molecule is present in the dermopharmaceutical or cosmetic composition in a concentration of 0.001-25% by weight.